Hypothesis

If colonic short‑chain fatty acid (SCFA) production drives vagal afferent signaling that directly modulates hippocampal DNA methylation patterns, then enhancing gut‑derived SCFA levels will lower brain epigenetic age independent of central nervous system‑targeted interventions.

Mechanistic Rationale

The gut microbiome fermenting dietary fiber yields acetate, propionate, and butyrate. These SCFAs activate enteroendocrine cells to release serotonin and peptide YY, which stimulate vagal afferents via 5‑HT3 and Y2 receptors. Vagal signaling reaches the nucleus tractus solitarius and, through the dorsal vagal complex, influences hippocampal neurogenesis via BDNF‑CREB cascades. Recent work shows that vagal afferents can alter histone acetylation in the hippocampus, but the link to DNA methylation remains untested. We propose that sustained SCFA‑vagal tone recruits TET enzymes to hippocampal promoters, driving demethylation of age‑associated CpG sites (e.g., those in the Foboxo3 and Sirt1 loci) as measured by murine epigenetic clocks.

Experimental Design

1. Animal model: 20‑month‑old C57BL/6 mice (n=10 per group).
2. Interventions:
   • SCFA boost: 5% inulin‑type fructan diet (prebiotic) for 8 weeks.
   • Vagotomy control: subdiaphragmatic vagotomy + prebiotic.
   • Central OSK control: hippocampal‑specific AAV‑OSK expression (transient, 2‑week pulse) without dietary change.
   • Sham: normal diet, surgery sham.
3. Readouts (collected at baseline and week 8):
   • Fecal SCFA concentrations (GC‑MS).
   • Vagal firing rate (in vivo electrophysiology of the cervical vagus).
   • Hippocampal methylation age using the mouse Horvath clock (targeted bisulfite sequencing of CpG sites).
   • Cognitive performance (Morris water maze, novel object recognition).
   • Hippocampal BDNF and TET2 expression (qPCR, Western blot).

Predictions

• Primary: Prebiotic‑fed intact mice will show a significant reduction (~1.5 years) in hippocampal methylation age relative to sham, accompanied by improved memory.
• Vagotomy: Despite elevated fecal SCFAs, vagotomized mice will not exhibit methylation age reversal, confirming vagal dependence.
• Central OSK: Hippocampal OSK will reset methylation age but will not improve memory unless paired with intact gut‑brain signaling, indicating that gut‑derived cues are necessary for functional outcomes.

Falsifiability

If prebiotic treatment fails to alter hippocampal methylation age in intact animals, or if vagotomy does not block the epigenetic effect despite confirmed SCFA elevation, the hypothesis is falsified. Similarly, if OSK‑induced methylation reset yields cognitive benefits irrespective of vagal integrity, the upstream role of gut signaling would be refuted.

Broader Impact

This study directly tests the directionality of the gut‑brain axis in aging using a quantitative epigenetic readout. A positive result would reorient longevity strategies toward microbiota‑targeted, vagal‑mediated interventions as a primary lever for brain rejuvenation, reshaping the design of combinatorial re‑programming protocols.